shRNA沉默HMGA2对食管癌ECA109细胞增殖和凋亡的影响

摘要

目的 探讨shRNA沉默HMGA2对食管癌ECA109细胞增殖、凋亡、迁移和侵袭的影响,并探究所涉及的相关机制.方法 构建针对人HMGA2基因的shRNA表达载体,瞬时转染至人食管癌细胞系ECA109中,24 h后应用蛋白免疫印迹法(WB)检测转染效率;应用MTT法比较转染后sh-HMGA2组与sh-NC组细胞增殖情况;应用流式细胞术检测HMGA2沉默对细胞凋亡的影响;应用Transwell测定评估HMGA2沉默对细胞迁移和侵袭能力的影响;应用WB检测HMGA2沉默对ECA109细胞内HMGA2、TGF-βRⅡ、Vimentin、Smad2、Smad3、磷酸化Smad2/3、E-cadherin、N-cadherin、Bax、Bcl-xl、MMP-2、MMP-9蛋白表达的影响.结果 与sh-NC组相比,sh-HM-GA2组中HMGA2的蛋白表达水平降低(P﹤0.05).MTT结果显示,与sh-NC组相比,sh-HMGA2组细胞增殖率明显降低(P﹤0.01).流式细胞术结果显示,与sh-NC组相比,sh-HMGA2组细胞凋亡率增加(P﹤0.05).Tran-swell测定结果显示,与sh-NC组相比,sh-HMGA2组中ECA109细胞迁移和侵袭数量均减少(P﹤0.05).WB结果显示,与sh-NC组相比,sh-HMGA2组细胞内Bax和E-cadherin表达水平增加,而Bcl-xl、Vimentin、N-cadherin、MMP-2、MMP-9、TGF-β、Smad2、Smad3、p-Smad2/3蛋白表达水平均降低(P﹤0.05).结论 shRNA沉默HMGA2基因通过下调Bcl-xl表达、上调Bax表达水平来抑制食管癌细胞系ECA109细胞增殖并促进细胞凋亡,并通过抑制MMP及上皮间质转化来抑制细胞迁移和侵袭,这一过程涉及TGF-β/Smad信号通路.%Objective To investigate the effect of shRNA-mediated silencing of HMGA2 on the proliferation, apopto-sis, migration and invasion of esophageal carcinoma ECA109 cells, and to explore the related mechanisms involved. Method The shRNA expression vector targeting human HMGA2 gene was constructed and transfected into human esophageal cancer cell line ECA109, and the transfection efficiency was detected by Western blot (WB) after 24 h. The proliferation of cells in sh-HMGA2 group and sh-NC group were compared by MTT assay. The effect of HMGA2 silenc-ing on apoptosis was detected by flow cytometry. Transwell was used to assess the effect of HMGA2 silencing on cell mi-gration and invasion. WB was used to detect the expression of HMGA2, TGF-βRII, Vimentin, Smad2, Smad3, phosphor-ylated Smad2/3, E-cadherin, N-cadherin, Bax, Bcl-xl, MMP-2 and MMP-9 in ECA109 cells. Result Compared with sh-NC, the expression of HMGA2 protein in sh-HMGA2 cells was significantly decreased (P<0.05). MTT results showed that the cell proliferation of sh-HMGA2 group was significantly lower than that of sh-NC group (P<0.01). Flow cytome-try showed that the apoptotic rate of sh-HMGA2 group was significantly higher compared with sh-NC group (P<0.05). Transwell assay showed that migration and invasion of ECA109 cells in sh-HMGA2 group were significantly lower than those in sh-NC group (P<0.05). WB showed that, the expression of Bax and E-cadherin in sh-HMGA2 group was signifi-cantly higher than that in sh-NC group, while Bcl-xl, Vimentin, N-cadherin, MMP-2, MMP-9, TGF-β, Smad2, Smad3, p-Smad2/3 protein expression levels were significantly reduced (P<0.05). Conclusion shRNA-mediated silencing of HMGA2 down-regulated the expression of Bcl-xl while up-regulating Bax to inhibit the proliferation and promote apopto-sis of esophageal cancer cell line ECA109 cells, and inhibit the migration and invasion by inhibiting MMP and EMT, this process involves TGF-β/Smad signaling pathway.